C/EBPs诱饵寡核苷酸对脓毒症小鼠的保护作用及机制研究

It is generally acknowledged that serious immunological defects and disorders occurred in sepsis. Strategies targeting multiple factors to inhibit the hyper-inflammatory response at the early phase and rebuild the immune balance at the late phase of sepsis provide hope for potential therapies in sepsis. Previous studies and our recent research provide evidence that the family of transcription factors C/EBPs plays a crucial role in the development of immune and inflammatory responses. However, yet there has hardly any reports showing that the C/EBPs could be used as an intervention target for sepsis treatment. Bioinformatics analysis showed that there are a few specificity recognition sequence of C/EBPs in the promoter region of several immune and inflammatory related genes including IL-1β、IL-6、NF-κB、PD-1 and PD-L1. Based on the above research background, we speculate that C/EBPs might be a potential intervention target for sepsis, inhibiting the DNA binding activity of C/EBPs could regulate expression of multiple immune and inflammation related target genes, and thereby provide protective effect against sepsis. According to the hypothesis, the expression, distribution and DNA binding activity of C/EBPs during inflammation and sepsis would be observed first; then a dumb-bell decoy oligonucleotides against C/EBPs (C/EBPs decoy ODNs) is synthesized according to the transcription factor decoy strategy, and the effect of C/EBPs decoy ODNs on inflammatory and immune responses, the multiple organ damage and survival of septic mice and its regulation on the expression of multiple immune and inflammation related target genes are investigated. It’s a novel study to explore the protective mechanism of targeting C/EBPs from two important aspects of sepsis treatment including inhibiting inflammation and regulating immune function and it might lay the laboratory foundation for sepsis treatment.

脓毒症时机体处于复杂的免疫紊乱状态，抑制早期炎症反应及晚期重建免疫平衡的多靶点干预有良好治疗前景。文献报道及申请者前期发现C/EBPs是一族在免疫炎症反应中起重要作用的转录因子，但其能否作为脓毒症干预靶点，国内外罕见报道。生物信息学分析表明免疫炎症相关基因IL-1β、IL-6、NF-κB、PD-1、PD-L1启动子区存在数个完整的C/EBPs特异性识别序列。基于上述背景，本项目提出假说：C/EBPs是脓毒症潜在干预靶点，干扰其 DNA结合活性可调控相关靶基因表达，多靶点调节脓毒症免疫炎症失衡，从而保护脓毒症。因此，本项目拟观察炎症及脓毒症时C/EBPs成员的表达、分布及DNA结合活性；并合成针对C/EBPs的诱饵寡脱氧核苷酸，在细胞及动物水平研究其对脓毒症小鼠炎症与免疫反应、多器官损伤及存活率的影响，观察其对炎症及免疫相关基因的表达调控，从抗炎及调节免疫功能两方面探讨其对脓毒症的保护机制。

脓毒症时机体处于复杂的免疫紊乱及失衡状态，重建其免疫稳态、联合多靶点免疫调节有良好应用前景。C/EBPs是一族在免疫炎症反应中起重要作用的转录因子，但其能否作为脓毒症干预靶点，目前并不清楚。生物信息学分析表明众多免疫炎症相关基因如IL-1β、IL-6、NF-κB、PD-1、PD-L1等启动子区存在数个完整的C/EBPs特异性识别序列。因此，为了寻找新的脓毒症防治靶点，本研究合成针对C/EBPs的诱饵寡核苷酸，在细胞及动物水平研究其对脓毒症小鼠炎症与免疫反应、多器官损伤及存活率的影响，观察其对相关基因的表达调控，从抗炎及调节免疫功能两方面探讨其对脓毒症的保护机制。结果发现合成的诱饵寡核苷酸（C/EBPs decoy ODNs）和对照核苷酸（mutant ODNs）满足后续实验要求。细胞实验发现，LPS所致的RAW264.7细胞中C/EBPs多个成员蛋白表达上升且DNA结合活性增强；C/EBPs decoy ODNs和mutant ODNs转染效率高且无明显细胞毒性作用，且不影响CEBPs蛋白表达。C/EBPs decoy ODNs能抑制LPS刺激下RAW264.7细胞TNF-α, IL-1β，IL-6, IL-18, MCP-1, MIF, caspase-1, NF-κB 1, ICAM 1, VCAM 1和Fgα的mRNA表达。动物实验发现C/EBPs decoy ODNs可显著改善脓毒症小鼠存活率，并呈剂量和时间依赖趋势；C/EBPs decoy ODNs可降低器官功能相关的酶学指标水平，减轻其心、肝、肺、肾的组织损伤；C/EBPs decoy ODNs降低早期脓毒症小鼠肺组织炎症相关因子TNF-α, IL-1β，IL-6, IL-18, MCP-1, MIF, caspase-1, NF-κB 1, ICAM 1, VCAM 1和Fgα的mRNA表达；C/EBPs decoy ODNs可抑制晚期脓毒症小鼠脾脏T淋巴细胞凋亡、降低Treg数目、抑制Treg表面功能相关分子PD-1及PD-L1的表达、上调Th1型细胞因子表达、下调Th2型细胞因子表达。以上发现表明C/EBPs可作为脓毒症防治的潜在干预靶点；C/EBPs decoy ODNs 通过干扰C/EBPs DNA 结合活性而调控免疫及炎症相关靶基因的表达，重建免疫平衡，发挥对脓毒症的保护作用。